Collapsin response mediator proteins (CRMPs) belong to a family of phosphoproteins, which mediate semaphorin/collapsin-induced growth cone collapse and are believed to be involved in both axonal guidance and neuronal differentiation. CRMPs are expressed mainly in the nervous system, especially during embryogenesis. Immunocytochemical studies have shown that CRMPs are distributed in the lamellipodia and filopodia of the growth cone, the shaft of axons, and the neuronal cell body. Their expression and phosphorylation are spatially and temporally regulated during development although their molecular mechanisms of action are yet to be clearly.
The members of CRMPs bear the sequence homology to UNC-33, a nematode protein, whose absence produces aberrant elongation of axons and uncoordinated movement in the worm Caenorhabditis elegans. (Li et al., Genetics (1992), 132(3): 675-689). CRMP family members have a 50%-70% amino acid sequence homology. Five members of the CRMP gene family (crmp-1, crmp-2, crmp-3, crmp-4, and crmp-5), encoding closely related 60-66 kDa proteins, have been independently cloned by various laboratories. Each CRMP is believed to have a unique function. The members of the CRMP family have been referred to as CRMP (collapsin response mediator protein), TOAD-64 (turned on after division of a 64 kD protein), Ulip (UNC-33 like phosphoprotein), DRP (dihydropyrimidinase related protein) and TUC (TOAD/Ulip/CRMP). Nonetheless, the most frequently used name in medical literature is CRMP.
Transcription of the CRMP gene is differentially regulated. (Kato et al., Histochem. Cell Biol. (2000), 97(11):6212-6217; Matsuo et al., J. Biol. Chem. (2000), 275(22):16560-16568; Quach et al., Gene (2000), 242(1-2): 175-182). Mouse CRMP-1, CRMP-4 and CRMP-5 are mainly expressed in the fetal brain and not in the brain of the adult mice. On the other hand, CRMP-2 and CRMP-3 are expressed in the brain of both the fetal and the adult mice. However, in the adult mice, CRMP-3 is localized in the cerebellum. In PC-12 cells, after induction of neuronal differentiation by nerve growth factor (NGF), CRMP-4 was strongly up-regulated, whereas CRMP-1 and CRMP-2 only increased slightly and CRMP-3 was down-regulated. (Byk et al., Eur. J. Biochem. (1998), 254(1): 14-24). At this time, only the promoter of human CRMP-4 has been isolated and analyzed. (Matsuo et al., J. Biol. Chem. (2000), 275(22): 16560-16568). No studies of the regulatory elements of other members of the CRMP family have been conducted.
Collapsin response mediator protein-1 (CRMP-1), also named as dihydropyrimidinase related protein-1 (DRP-1), is a 62 kDa phosphoprotein. CRMP-1 was originally discovered in the brain tissue and thought to be a brain specific protein involved in the collapsin-induced growth cone collapse during neural development. (Torres et al., DNA Res. (1998), 5(6): 393-395).
Recently, the inventors of the present invention discovered that the level of expression of the gene encoding CRMP-1 (hereinafter “CRMP-1 gene”) inversely affects cancer invasion and metastasis, (i.e., the higher the level of expression, the lower the incidence of cancer invasion and metastasis) and thus characterized the CRMP-1 gene as an invasion-suppression gene. (Shih et al., J. Natl. Cancer Inst. (2001), 93(18): 1392-1400; Chu et al., Am. J. Respir. Cell Mol. Biol. (1997), 17: 353-360; and Shih et al., Clinical & Exper. Metastasis (2003) 20: 69-76). The contents of these articles are herein incorporated by reference. The inventors of the present invention found that low-expression patients of CRMP-1 had more advanced diseases and lymph node metastases, while high-expression patients of CRMP-1 had a significantly longer disease-free and overall survival period.
In the invention to be presented in the following sections, the findings of the nucleic acid regulatory elements/sequences associated with the CRMP-1 gene are disclosed. These nucleic acid regulatory elements/sequences include, but are not limited to, the promoter, the basal transcription regulatory region, and the transcription factor binding sites which are located upstream of, and are operatively linked to, the CRMP-1 gene. Understanding the regulation of the expression of the CRMP-1 gene will lead to new strategies for treatment of cancer patients so as to halt cancer metastasis.